The specificities, influencing factors, and medical implications of bone circadian rhythms.

Physiological processes within the human body are regulated in approximately 24-h cycles known as circadian rhythms, serving to adapt to environmental changes. Bone rhythms play pivotal roles in bone development, metabolism, mineralization, and remodeling processes. Bone rhythms exhibit cell specificity, and different cells in bone display various expressions of clock genes. Multiple environmental factors, including light, feeding, exercise, and temperature, affect bone diurnal rhythms through the sympathetic nervous system and various hormones. Disruptions in bone diurnal rhythms contribute to the onset of skeletal disorders such as osteoporosis, osteoarthritis and skeletal hypoplasia. Conversely, these bone diseases can be effectively treated when aimed at the circadian clock in bone cells, including the rhythmic expressions of clock genes and drug targets. In this review, we describe the unique circadian rhythms in physiological activities of various bone cells. Then we summarize the factors synchronizing the diurnal rhythms of bone with the underlying mechanisms. Based on the review, we aim to build an overall understanding of the diurnal rhythms in bone and summarize the new preventive and therapeutic strategies for bone disorders.

BMAL1-Downregulation Aggravates Porphyromonas Gingivalis-Induced Atherosclerosis by Encouraging Oxidative Stress.

RATIONALE: Atherosclerotic cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerotic cardiovascular diseases are considered as chronic inflammation processes. In addition to risk factors associated with the cardiovascular system itself, pathogenic bacteria such as the periodontitis-associated Porphyromonas gingivalis (P gingivalis) are also closely correlated with the development of atherosclerosis, but the underlying mechanisms are still elusive. OBJECTIVE: To elucidate the mechanisms of P gingivalis-accelerated atherosclerosis and explore novel therapeutic strategies of atherosclerotic cardiovascular diseases. METHODS AND RESULTS: Bmal1-/- (brain and muscle Arnt-like protein 1) mice, ApoE-/- mice, Bmal1-/-ApoE-/- mice, conditional endothelial cell Bmal1 knockout mice (Bmal1fl/fl; Tek-Cre mice), and the corresponding jet-legged mouse model were used. Pgingivalis accelerates atherosclerosis progression by triggering arterial oxidative stress and inflammatory responses in ApoE-/- mice, accompanied by the perturbed circadian clock. Circadian clock disruption boosts P gingivalis-induced atherosclerosis progression. The mechanistic dissection shows that P gingivalis infection activates the TLRs-NF-κB signaling axis, which subsequently recruits DNMT-1 to methylate the BMAL1 promoter and thus suppresses BMAL1 transcription. The downregulation of BMAL1 releases CLOCK, which phosphorylates p65 and further enhances NF-κB signaling, elevating oxidative stress and inflammatory response in human aortic endothelial cells. Besides, the mouse model exhibits that joint administration of metronidazole and melatonin serves as an effective strategy for treating atherosclerotic cardiovascular diseases. CONCLUSIONS: P gingivalis accelerates atherosclerosis via the NF-κB-BMAL1-NF-κB signaling loop. Melatonin and metronidazole are promising auxiliary medications toward atherosclerotic cardiovascular diseases.

Circadian rhythms affect bone reconstruction by regulating bone energy metabolism.

Metabolism is one of the most complex cellular biochemical reactions, providing energy and substances for basic activities such as cell growth and proliferation. Early studies have shown that glucose is an important nutrient in osteoblasts. In addition, amino acid metabolism and fat metabolism also play important roles in bone reconstruction. Mammalian circadian clocks regulate the circadian cycles of various physiological functions. In vertebrates, circadian rhythms are mediated by a set of central clock genes: muscle and brain ARNT like-1 (Bmal1), muscle and brain ARNT like-2 (Bmal2), circadian rhythmic motion output cycle stagnates (Clock), cryptochrome 1 (Cry1), cryptochrome2 (Cry2), period 1 (Per1), period 2 (Per2), period 3 (Per3) and neuronal PAS domain protein 2 (Npas2). Negative feedback loops, controlled at both the transcriptional and posttranslational levels, adjust these clock genes in a diurnal manner. According to the results of studies on circadian transcriptomic studies in several tissues, most rhythmic genes are expressed in a tissue-specific manner and are affected by tissue-specific circadian rhythms. The circadian rhythm regulates several activities, including energy metabolism, feeding time, sleeping, and endocrine and immune functions. It has been reported that the circadian rhythms of mammals are closely related to bone metabolism. In this review, we discuss the regulation of the circadian rhythm/circadian clock gene in osteoblasts/osteoclasts and the energy metabolism of bone, and the relationship between circadian rhythm, bone remodeling, and energy metabolism. We also discuss the therapeutic potential of regulating circadian rhythms or changing energy metabolism on bone development/bone regeneration.

Single-Cell Genomics: Approaches and Utility in Immunology.

Single-cell genomics offers powerful tools for studying immune cells, which make it possible to observe rare and intermediate cell states that cannot be resolved at the population level. Advances in computer science and single-cell sequencing technology have created a data-driven revolution in immunology. The challenge for immunologists is to harness computing and turn an avalanche of quantitative data into meaningful discovery of immunological principles, predictive models, and strategies for therapeutics. Here, we review the current literature on computational analysis of single-cell RNA-sequencing data and discuss underlying assumptions, methods, and applications in immunology, and highlight important directions for future research.

BASIC: BCR assembly from single cells.

Motivation: The B-cell receptor enables individual B cells to identify diverse antigens, including bacterial and viral proteins. While advances in RNA-sequencing (RNA-seq) have enabled high throughput profiling of transcript expression in single cells, the unique task of assembling the full-length heavy and light chain sequences from single cell RNA-seq (scRNA-seq) in B cells has been largely unstudied. Results: We developed a new software tool, BASIC, which allows investigators to use scRNA-seq for assembling BCR sequences at single-cell resolution. To demonstrate the utility of our software, we subjected nearly 200 single human B cells to scRNA-seq, assembled the full-length heavy and the light chains, and experimentally confirmed these results by using single-cell primer-based nested PCRs and Sanger sequencing. Availability and Implementation: http://ttic.uchicago.edu/∼aakhan/BASIC Contact: aakhan@ttic.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

DeepCNF-D: Predicting Protein Order/Disorder Regions by Weighted Deep Convolutional Neural Fields.

Intrinsically disordered proteins or protein regions are involved in key biological processes including regulation of transcription, signal transduction, and alternative splicing. Accurately predicting order/disorder regions ab initio from the protein sequence is a prerequisite step for further analysis of functions and mechanisms for these disordered regions. This work presents a learning method, weighted DeepCNF (Deep Convolutional Neural Fields), to improve the accuracy of order/disorder prediction by exploiting the long-range sequential information and the interdependency between adjacent order/disorder labels and by assigning different weights for each label during training and prediction to solve the label imbalance issue. Evaluated by the CASP9 and CASP10 targets, our method obtains 0.855 and 0.898 AUC values, which are higher than the state-of-the-art single ab initio predictors.

Transcriptome comparison reveals the patterns of selection in domesticated and wild ramie (Boehmeria nivea L. Gaud).

Ramie is an old fiber crop, cultivated for thousands of years in China. The cultivar ramie evolved from the wild species Qingyezhuma (QYZM, Boehmeria nivea var. tenacissima). However, the mechanism of domestication of this old fiber crop is poorly understood. In order to characterize the selective pattern in ramie domestication, orthologous genes between the transcriptomes of domesticated ramie variety Zhongzhu 1 (ZZ1) and wild QYZM were assessed using bidirectional best-hit method and ratio of non-synonymous (Ka) to synonymous (Ks) nucleotide substitutions was estimated. Sequence comparison of 56,932 and 59,246 unigenes from the wild QYZM and domesticated ZZ1, respectively, helped identify 10,745 orthologous unigene pairs with a total orthologous length of 10.18 Mb. Among these unigenes, 85 and 13 genes were found to undergo significant purifying and positive selection, respectively. Most of the selected genes were homologs of those involved in abiotic stress tolerance or disease resistance in other plants, suggesting that abiotic and biotic stresses were important selective pressures in ramie domestication. Two genes probably related to the fiber yield of ramie were subjected to positive selection, which may be caused by human manipulation. Thus, our results show the pervasive effects of artificial and natural selections on the accelerated domestication of ramie from its wild relative.

Identification of 32 full-length NAC transcription factors in ramie (Boehmeria nivea L. Gaud) and characterization of the expression pattern of these genes.

NAM, ATAF, and CUC (NAC) genes are plant-specific transcription factors (TFs) that play key roles in plant growth, development, and stress tolerance. To date, none of the ramie NAC (BnNAC) genes had been identified, even though ramie is one of the most important natural fiber crops. In order to mine the BnNAC TFs and identify their potential function, the search for BnNAC genes against two pools of unigenes de novo assembled from the RNA-seq in our two previous studies was performed, and a total of 32 full-length BnNAC genes were identified in this study. Forty-seven function-known NAC proteins published in other species, in concert with these 32 BnNAC proteins were subjected to phylogenetic analysis, and the result showed that all the 79 NAC proteins can be divided into eight groups (NAC-I-VIII). Among the 32 BnNAC genes, 24, 2, and 1 gene showed higher expression in stem xylem, leaf, and flower, respectively. Furthermore, the expression of 14, 11 and 4 BnNAC genes was regulated by drought, cadmium stress, and infection by root lesion nematode, respectively. Interestingly, there were five BnNAC TFs which showed high homology with the NAC TFs of other species involved in regulating the secondary wall synthesis, and their expressions were not regulated by drought and cadmium stress. These results suggested that the BnNAC family might have a functional diversity. The identification of these 32 full-length BnNAC genes and the characterization of their expression pattern provide a basis for future clarification of their functions in ramie growth and development.

Application of Antioxidant Compounds in Bone Defect Repair.

Bone defects caused by trauma, tumor resection, and infections are significant clinical challenges. Excessive reactive oxygen species (ROS) usually accumulate in the defect area, which may impair the function of cells involved in bone formation, posing a serious challenge for bone repair. Due to the potent ROS scavenging ability, as well as potential anti-inflammatory and immunomodulatory activities, antioxidants play an indispensable role in the maintenance and protection of bone health and have gained increasing attention in recent years. This narrative review aims to give an overview of the main research directions on the application of antioxidant compounds in bone defect repair over the past decade. In addition, the positive effects of various antioxidants and their biomaterial delivery systems in bone repair are summarized to provide new insights for exploring antioxidant-based strategies for bone defect repair.

Time of exercise differentially impacts bone growth in mice.

Although physical training has been shown to improve bone mass, the time of day to exercise for optimal bone growth remains uncertain. Here we show that engaging in physical activity during the early active phase, as opposed to the subsequent active or rest phase, results in a more substantial increase in bone length of male and female mice. Transcriptomic and metabolomic methodologies identify that exercise during the early active phase significantly upregulates genes associated with bone development and metabolism. Notably, oxidative phosphorylation-related genes show a rhythmic expression in the chondrification centre, with a peak at the early active phase, when more rhythmic genes in bone metabolism are expressed and bone growth is synergistically promoted by affecting oxidative phosphorylation, which is confirmed by subsequent pharmacological investigations. Finally, we construct a signalling network to predict the impact of exercise on bone growth. Collectively, our research sheds light on the intricacies of human exercise physiology, offering valuable implications for interventions.

Osteocyte ferroptosis induced by ATF3/TFR1 contributes to cortical bone loss during ageing.

Cortical bone loss is intricately associated with ageing and coincides with iron accumulation. The precise role of ferroptosis, characterized by iron overload and lipid peroxidation, in senescent osteocytes remains elusive. We found that ferroptosis was a crucial mode of osteocyte death in cortical bone during ageing. Using a single-cell transcriptome analysis, we identified activating transcription factor 3 (ATF3) as a critical driver of osteocyte ferroptosis. Elevated ATF3 expression in senescent osteocytes promotes iron uptake by upregulating transferrin receptor 1 while simultaneously inhibiting solute carrier family 7-member 11-mediated cystine import. This process leads to an iron overload and lipid peroxidation, culminating in ferroptosis. Importantly, ATF3 inhibition in aged mice effectively alleviated ferroptosis in the cortical bone and mitigated cortical bone mass loss. Taken together, our findings establish a pivotal role of ferroptosis in cortical bone loss in older adults, providing promising prevention and treatment strategies for osteoporosis and fractures.

De novo assembly and characterization of transcriptome using Illumina paired-end sequencing and identification of CesA gene in ramie (Boehmeria nivea L. Gaud).

BACKGROUND: Ramie fiber, extracted from vegetative organ stem bast, is one of the most important natural fibers. Understanding the molecular mechanisms of the vegetative growth of the ramie and the formation and development of bast fiber is essential for improving the yield and quality of the ramie fiber. However, only 418 expressed tag sequences (ESTs) of ramie deposited in public databases are far from sufficient to understand the molecular mechanisms. Thus, high-throughput transcriptome sequencing is essential to generate enormous ramie transcript sequences for the purpose of gene discovery, especially genes such as the cellulose synthase (CesA) gene. RESULTS: Using Illumina paired-end sequencing, about 53 million sequencing reads were generated. De novo assembly yielded 43,990 unigenes with an average length of 824 bp. By sequence similarity searching for known proteins, a total of 34,192 (77.7%) genes were annotated for their function. Out of these annotated unigenes, 16,050 and 13,042 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 19,846 unigenes were mapped to 126 KEGG pathways, and 565 genes were assigned to http://starch and sucrose metabolic pathway which was related with cellulose biosynthesis. Additionally, 51 CesA genes involved in cellulose biosynthesis were identified. Analysis of tissue-specific expression pattern of the 51 CesA genes revealed that there were 36 genes with a relatively high expression levels in the stem bark, which suggests that they are most likely responsible for the biosynthesis of bast fiber. CONCLUSION: To the best of our knowledge, this study is the first to characterize the ramie transcriptome and the substantial amount of transcripts obtained will accelerate the understanding of the ramie vegetative growth and development mechanism. Moreover, discovery of the 36 CesA genes with relatively high expression levels in the stem bark will present an opportunity to understand the ramie bast fiber formation and development mechanisms.

Identification of drought stress-responsive transcription factors in ramie (Boehmeria nivea L. Gaud).

BACKGROUND: Ramie fiber extracted from stem bark is one of the most important natural fibers. Drought is a main environment stress which severely inhibits the stem growth of ramie and leads to a decrease of the fiber yield. The drought stress-regulatory mechanism of ramie is poorly understood. RESULT: Using Illumina sequencing, approximately 4.8 and 4.7 million (M) 21-nt cDNA tags were respectively sequenced in the cDNA libraries derived from the drought-stressed ramie (DS) and the control ramie under well water condition (CO). The tags generated from the two libraries were aligned with ramie transcriptome to annotate their function and a total of 23,912 and 22,826 ramie genes were matched by these tags of DS and CO library, respectively. Comparison of gene expression level between CO and DS ramie based on the differences of tag frequencies appearing in the two libraries revealed that there were 1516 potential drought stress-responsive genes, in which 24 genes function as transcription factor (TF). Among these 24 TFs, the unigene19721 encoding the DELLA protein which is a key negative regulator in gibberellins (GAs) signal pathway was probably markedly up-regulated under water stress for a increase of tag abundance in DS library, which is possibly responsible for the inhibition of the growth of drought-stressed ramie. In order to validate the change of expression of these potential stress-responsive TFs under water deficit condition, the unigene19721 and another eleven potential stress-responsive TFs were chosen for further expression analysis in well-watered and drought-stressed ramie by real-time quantitative PCR (qRT-PCR) and the result showed that all 12 TFs were authentically involved in the response of drought stress. CONCLUSION: In this study, twelve TFs involving in the response of drought stress were first found by Illumina tag-sequencing and qRT-PCR in ramie. The discovery of these drought stress-responsive TFs will be helpful for further understanding the drought stress-regulatory mechanism of ramie and improving the drought tolerance ability of ramie.

Interaction between apical periodontitis and systemic disease (Review).

Apical periodontitis is an oral common inflammatory disease initiated by infection of pulp chamber and is characterized by destruction and resorption of the periapical bone. As a local infection, pathogens and their products in periapical tissues, as well as inflammatory cytokines produced in periapical lesions, enter the blood circulation, triggering systemic immune responses and leading to the pathogenesis of various types of systemic disease. Therefore, apical periodontitis might be associated with systemic disease rather than solely simple local oral disease. In addition, the existence of a hyperinflammatory state in certain patients with chronic inflammation­related disorder may affect the progression or prognosis of apical periodontitis. However, the association and potential mechanisms between apical periodontitis and systemic diseases remain unclear. An in­depth understanding of the association between apical periodontitis and systemic disease will be useful for both dentists and physicians to eliminate the possible risk factors and promote the healing of apical periodontitis and systemic disease. Thus, the aim of the present review is to introduce the potential relationship between apical periodontitis and systemic disease.

Targeting RORα in macrophages to boost diabetic bone regeneration.

Diabetes mellitus (DM) has become a serious threat to human health. Bone regeneration deficiency and nonunion caused by DM is perceived as a worldwide epidemic, with a very high socioeconomic impact on public health. Here, we find that targeted activation of retinoic acid-related orphan receptor α (RORα) by SR1078 in the early stage of bone defect repair can significantly promote in situ bone regeneration of DM rats. Bone regeneration relies on the activation of macrophage RORα in the early bone repair, but RORα of DM rats fails to upregulation as hyperglycemic inflammatory microenvironment induced IGF1-AMPK signalling deficiency. Mechanistic investigations suggest that RORα is vital for macrophage-induced migration and proliferation of bone mesenchymal stem cells (BMSCs) via a CCL3/IL-6 depending manner. In summary, our study identifies RORα expressed in macrophages during the early stage of bone defect repair is crucial for in situ bone regeneration, and offers a novel strategy for bone regeneration therapy and fracture repair in DM patients.

Developmental growth plate cartilage formation suppressed by artificial light at night via inhibiting BMAL1-driven collagen hydroxylation.

Exposure to artificial light at night (LAN) can induce obesity, depressive disorder and osteoporosis, but the pernicious effects of excessive LAN exposure on tissue structure are poorly understood. Here, we demonstrated that artificial LAN can impair developmental growth plate cartilage extracellular matrix (ECM) formation and cause endoplasmic reticulum (ER) dilation, which in turn compromises bone formation. Excessive LAN exposure induces downregulation of the core circadian clock protein BMAL1, which leads to collagen accumulation in the ER. Further investigations suggest that BMAL1 is the direct transcriptional activator of prolyl 4-hydroxylase subunit alpha 1 (P4ha1) in chondrocytes, which orchestrates collagen prolyl hydroxylation and secretion. BMAL1 downregulation induced by LAN markedly inhibits proline hydroxylation and transport of collagen from ER to golgi, thereby inducing ER stress in chondrocytes. Restoration of BMAL1/P4HA1 signaling can effectively rescue the dysregulation of cartilage formation within the developmental growth plate induced by artificial LAN exposure. In summary, our investigations suggested that LAN is a significant risk factor in bone growth and development, and a proposed novel strategy targeting enhancement of BMAL1-mediated collagen hydroxylation could be a potential therapeutic approach to facilitate bone growth.

F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production.

BACKGROUND: Tumor-resident microbiota has been documented for various cancer types. Oral squamous cell carcinoma (OSCC) is also enriched with microbiota, while the significance of microbiota in shaping the OSCC microenvironment remains elusive. METHODS: We used bioinformatics and clinical sample analysis to explore relationship between F. nucleatum and OSCC progression. Xenograft tumor model, metabolic screening and RNA sequencing were performed to elucidate mechanisms of pro-tumor role of F. nucleatum. FINDINGS: We show that a major protumorigenic bacterium, F. nucleatum, accumulates in invasive margins of OSCC tissues and drives tumor-associated macrophages (TAMs) formation. The mechanistic dissection shows that OSCC-resident F. nucleatum triggers the GalNAc-Autophagy-TBC1D5 signaling, leading to GLUT1 aggregation in the plasma membrane and the deposition of extracellular lactate. Simultaneous functional inhibition of GalNAc and GLUT1 efficiently reduces TAMs formation and restrains OSCC progression. INTERPRETATION: These findings suggest that tumor-resident microbiota affects the immunomodulatory and protumorigenic microenvironment via modulating glycolysis and extracellular lactate deposition. The targeted intervention of this process could provide a distinct clinical strategy for patients with advanced OSCC. FUNDING: This work was supported by the National Natural Science Foundation of China for Key Program Projects (82030070, to LC) and Distinguished Young Scholars (31725011, to LC), as well as Innovation Team Project of Hubei Province (2020CFA014, to LC).

Hormone and implant osseointegration: Elaboration of the relationship among function, preclinical, and clinical practice.

As clusters of peptides or steroids capable of high-efficiency information transmission, hormones have been substantiated to coordinate metabolism, growth, development, and other physiological processes, especially in bone physiology and repair metabolism. In recent years, the application of hormones for implant osseointegration has become a research hotspot. Herein, we provide a comprehensive overview of the relevant reports on endogenous hormones and their corresponding supplementary preparations to explore the association between hormones and the prognosis of implants. We also discuss the effects and mechanisms of insulin, parathyroid hormone, melatonin, vitamin D, and growth hormone on osseointegration at the molecular and body levels to provide a foothold and guide future research on the systemic conditions that affect the implantation process and expand the relative contraindications of the implant, and the pre-and post-operative precautions. This review shows that systemic hormones can regulate the osseointegration of oral implants through endogenous or exogenous drug-delivery methods.

Dysfunction of macrophages leads to diabetic bone regeneration deficiency.

Insufficient bone matrix formation caused by diabetic chronic inflammation can result in bone nonunion, which is perceived as a worldwide epidemic, with a substantial socioeconomic and public health burden. Macrophages in microenvironment orchestrate the inflammation and launch the process of bone remodeling and repair, but aberrant activation of macrophages can drive drastic inflammatory responses during diabetic bone regeneration. In diabetes mellitus, the proliferation of resident macrophages in bone microenvironment is limited, while enhanced myeloid differentiation of hematopoietic stem cells (HSCs) leads to increased and constant monocyte recruitment and thus macrophages shift toward the classic pro-inflammatory phenotype, which leads to the deficiency of bone regeneration. In this review, we systematically summarized the anomalous origin of macrophages under diabetic conditions. Moreover, we evaluated the deficit of pro-regeneration macrophages in the diabetic inflammatory microenvironment. Finally, we further discussed the latest developments on strategies based on targeting macrophages to promote diabetic bone regeneration. Briefly, this review aimed to provide a basis for modulating the biological functions of macrophages to accelerate bone regeneration and rescue diabetic fracture healing in the future.

Transcriptome analysis provides new insights into cold adaptation of corsac fox (Vulpes Corsac).

Vulpesare widely distributed throughout the world and have undergone drastic physiological and phenotypic changes in response to their environment. However, little is known about the underlying genetic causes of these traits, especially Vulpes corsac. In this study, RNA-Seq was used to obtain a comprehensive dataset for multiple pooled tissues of corsac fox, and selection analysis of orthologous genes was performed to identify the genes that may be influenced by the low-temperature environment. More than 6.32 Gb clean reads were obtained and assembled into a total of 173,353 unigenes with an average length of 557 bp for corsac fox. Selective pressure analysis showed that 16 positively selected genes (PSGs) were identified in corsac fox, red fox, and arctic fox. Enrichment analysis of PSGs showed that the LRP11 gene was enriched in several pathways related to the low-temperature response and might play a key role in response to environmental stimuli of foxes. In addition, several positively selected genes were related to DNA damage repair (ELP2 and CHAF1A), innate immunity (ARRDC4 and S100A12), and the respiratory chain (NDUFA5), and these positively selected genes might play a role in adaptation to harsh wild fox environments. The results of common orthologous gene analysis showed that gene flow or convergent evolution might be an important factor in promoting regional differentiation of foxes. Our study provides a valuable transcriptomic resource for the evolutionary history of the corsac fox and the adaptations to the extreme environments.